Evaluation of the Effects of Whey Protein and Moringa Oleifera Leaves Extract Mixture on Osseointegration in Rabbits

Background: Osteogenic, antioxidant and anti-inflammatory effects of Whey protein and M. oleifera gel prompted us to evaluate their role alone or in combination on osseointegration in rabbits. Methods: In this study, 24 titanium implants were inserted in the femurs of six rabbits. One implant was placed without treatment, and another one was coated with a mixture of whey protein and M. oleifera gel for each side. The animals were divided into two groups of 2- and 6-week intervals and evaluated using histopathological and immunohistochemical techniques. Results: Histological evaluation revealed a significant difference between the experimental and the control groups after two weeks in osteoblast and osteocyte counts. The experimental group had mature bone development after six weeks of implantation, while the control group had a woven bone. Immunohistochemical results showed that the experimental group, compared to the control group, exhibited early positive expression of osteoblast cells at two weeks after the experiment. Based on histopathological observations, the experimental group showed a tiny area of collagenous fiber in 6th week after the implantation. Conclusion: A mixture of whey protein and M. oleifera could accelerate osseointegration and healing processes.


INTRODUCTION
sseointegration, the direct contact between implant and bone surfaces, frequently occurs in two dimensions.The proportion of bone-toimplant contact and the pressure required to dislodge the implant from the bone act as a biological readout for osseointegration [1] .For dental implants, which predominantly consist of titanium, osseointegration is crucial.Surface etching and sandblasting of titaniumbased dental implants can induce rough surfaces, which appear to be ideal for osseointegration [2] .
Cow's milk is a rich source of a premium protein known as whey protein.Casein and whey proteins comprise around 80% and 20% of the protein components of the milk, respectively [3] .Whey has a protein content of less than 1% that mostly consists of lactoglobulin and lactalbumin.It also contains small amounts of glycoproteins such as lanolin, lactoferrin, transferrin, and lactoperoxidase, as well as comprises proteases, peptones, immunoglobulins, and bovine serum albumin [4] .Whey protein inhibits the disruptive effects of stress, as a part of its antioxidant action related to glutathione synthesis.Bioactive peptides are released from the whey proteins, leading to an increased intracellular glutathione level and decreased production of IL-8, a cytokine involved in respiratory diseases [5] .Considering that the whey protein includes various components with the ability to prevent the destructive activity of toxins, germs, and viruses, it can stimulate immune cells and/or prevent infection.Lactoferrin and its peptide derivatives, lactoperoxidase, lactoferricin, and sphingolipids, are a number of the components with antibacterial activity [6] .Lactoferrin, lanolin, and lactoglobulin have also shown an inhibitory effect against HIV type 1 [7] .β-lactoglobulin is an effective agent in preventing the spread of HIV and genital herpes virus infections.It can inhibit parasitic protozoa, fungi, yeast, Gram-positive and Gram-negative bacteria [8] .
The leaves, seeds, bark, roots, and flowers of the M. oleifera plant are widely used in traditional medicine, owing to their low side effects, safety to use, and strong antioxidant activity.Yet no adverse outcomes have been reported for this plant in human studies [9] .The leaves of M. oleifera consist of vitamins (A, B6, and C), iron, riboflavin, proteins, and magnesium; however, the antioxidant activity of the extract of this part of the plant has rarely been investigated.The M. oleifera also contains a flavonoid that has lately been advocated for the prevention and treatment of COVID-19 [10] .Flavonoids and bioactive substances found in M. oleifera, help protect people from chronic degenerative diseases.The main flavonoids of the plant are quercetin and kaempferol derivatives, which have antiinflammatory properties in the form of glycosides, malonyl glycosides, acetyl glycosides, and succinyl glycosides [11] .
Growth factors have active participation in the formation and regeneration of bones.These chemicals control osteoprogenitor cell migration, their proliferation and differentiation to mature osteoblasts, type I collagen production, and matrix apposition through autocrine and paracrine signaling processes [12] .IGF-I is important in the regulation of new bone formation.It is a small peptide with a similar structure to insulin that acts as a regulator of skeletal growth.IGF-I is synthesized in the liver under the effect of growth hormone and is bound to IGF-binding protein-3.Circulating IGF-I affects longitudinal bone growth.Osteoblasts can locally synthesize IGF-I in response to PTH, which mediates its anabolic effects on bone.IGF-I and -II are stored in the extracellular matrix of bone and serve as the most abundant growth factors [13] .
The present study was conducted to histologically, histochemically and immunohistochemically evaluate the effect of whey protein and M. oleifera gel combination on osseointegration.

Study samples
Six adult healthy male white New Zealand rabbits, aged between 9 and 13 months and weighing 2.5-3 kg, were used in the study.A total of four dental implants, two in the right femur and two in the left femur, were placed in each rabbit by surgical operation.One of the implants placed on each side was left untreated, and the other was covered with a mixture of 0.25 mg of whey protein and 0.25 ml of M. oleifera gel [14] .The gel was prepared by drying, hardening, and grinding the M. oleifera leaves [15] .

Surgical process and slide preparation
Intermittent drilling and continuous irrigation were applied by physiological saline to prevent heating of the surgical area.Then 1.8-mm diameter holes were created in the place of the implants, with the help of a micro motor fitted with a round bur.A 20-mm gap was left between the two holes.Tissue debris in the operation regions was removed by irrigating the area using physiological saline [16] .

Slide preparation
At the end of the 2 nd and 6 th weeks after implantation, three animals were sacrificed in each period.Bone tissue samples containing the implant site were processed through routine histotechniques, by immersing in paraffin blocks and then sectioning.The sections were stained by H&E and Masson's trichrome.The specimens were finally examined under a light microscope, and the regions surrounding the implants were evaluated histologically and histomorphometrically.Bone marrow and trabecular area were expressed in µm 2 , while the newly formed bone area was expressed as a percentage of the measured tissue area (1.45 mm 2 ) according to the following equation: newly formed bone area = measured bone area/measured tissue area (×100) [17] , using Image J software.Osteoblast and osteocyte counts were determined by considering the mean numbers in the bone tissue with dimensions of 1.41 × 1.05 mm located in the first three threads of the implant, under ×40 magnification [18,19] .For the immunohistochemical demonstration of IGF-I, a polyclonal primary antibody (Abcam, UK) was employed.Each sample was evaluated for the intensity of the brown cytoplasmic DAB reaction product.The intensity of stained cells scored from 0 to 3 as follows: (0) negative, (1) weak, (2) moderate, and (3) strong.The positive expression stained cells scored from 0-4, score (0) belonged to the frequency of positive cells less than 2%, score (1) was between 2 and 10%, score (2) between 11 and 25%, score (3) between 26 and 50%, and score (4) was greater than 50%, respectively.Total immunoreactivity was determined as the sum of the staining intensity and the percentage of immune-positive cells [20] .In Masson's trichrome-stained specimens, osteoid tissue was examined because the tissue was rich in type I collagenous fibers.Iran.Biomed.J. 28 (2 & 3): 82-89 Fig. 1.Sections of the defect sites in the study groups at the end of 2 nd week after implantation.(A) In the control group, areas between the threads of the implants are occupied mainly with loose connective tissue rich in capillary blood vessels and adipocytes.(B) In the experimental group, a narrow fibrocartilage tissue, with a small number of osteoblastic cells, in the areas adjacent to the implant threads was observed.The sections were observed under a light microscope with magnitudes ×200 and ×400.

Statistical analysis
The data were analyzed using the SPSS 25 software.The mean, median, standard deviations and p values of each parameter were determined.P values under or equal to 0.05 were considered statistically significant.

Histochemical findings
At the end of the 2 nd week following implantation, histological findings of the implant areas in the control group showed that the area between the implant threads was substantially filled with loose connective tissue rich in capillary blood vessels and fat cells.In addition, no broad fibrocartilage tissue was observed in the area (Fig. 1A).In contrast, in the experimental group, loose connective tissue such as fibrocartilage tissue consisting of osteoblastic and osteocytic cells, have mainly filled the spaces between the implant threads at the end of the 2 nd week (Fig. 1B).At the end of the 6 th week after implantation, the regions between implant threads were mostly filled with osseous tissue in both study groups (Fig. 2).However, the experimental animals (Fig. 2A) exhibited an advanced production of mature, trabecular bone relative to the control animals (Fig. 2B).In the same period, the experimental group had higher osteocyte cell number, newly formed bone rate, and trabecular area as compared to the control group.However, osteoblastic cell numbers and bone marrow area were lower than those of the control.Moreover, the experimental group had significantly (p < 0.01) higher values in all parameters, except for the bone marrow area, at the end of the 2nd week and Osteoblastes number at the 6th week (Table 1).Fig. 2. Sections of the defect sites in the study groups at the end of the 6 th week after implantation.The areas between the threads of the implant were filled with trabecular bone.(A) In the experimental group, bone is more organized, and trabeculae are greater than (B) the control group.The sections were observed under a light microscope with magnitudes ×200 and ×400.

IGF-I expression
Results of the immunohistochemical investigations showed that osteoblast, osteoclast, osteocytes, and bone marrow cells of the control group did not have immunepositive reaction to IGF-I at both periods of the experiment (Fig. 3).In the experimental group, most of the investigated cells displayed IGF-I immune reactivity, except for bone marrow stromal cells (Fig. 3A).Similar results were also observed at the end of the 6 th week after implantation.Immune-positive cell scores of the experimental group were significantly (p < 0.05) higher than those of the control group at the end of the 2 nd and 6 th weeks of the experiment (Table 2).

Findings of Masson's trichrome-staining
Results of histochemical findings showed that the two study groups had a sizable red-colored region that represented the amount of collagen in the second week (Fig. 4A).In the sixth week, the experimental group displayed a small region indicative of bone mineralization, whereas the control group exhibited a large area of collagen intensity (Fig. 4B).

DISCUSSION
Titanium and its alloys are the ideal bio-integrated materials for dental implants.The integration between the tissue and implants depends on bone quality and quantity, as well as implant material and loading conditions [21] .In the current study, a combination of M. oleifera and whey protein was used because the primary component of whey protein, namely lactoglobulin, can alter the immunity and acts as an antioxidant, anticancer, antiviral, and antibacterial agent [22] .Also, M. oleifera leaf extract is rich in tannins, saponins, alkaloids, and flavonoids, and it contains flavonoids that can promote osteoblast development and proliferation [23] .Interestingly, saponins influence osteogenic activity, thus promoting osteoblast proliferation, differentiation, and bone formation [24] .
At the end of the 2 nd week of the experiment, the histological analysis of the control group revealed that the regions between the screw threads were filled with loose connective tissue separated with a distinct line from the original bone.Furthermore, osteoclastic activity at the implant site was weak, and few bone   spicules were observed in the implant area.This observation has also been demonstrated by Othman and Al-Ghaban [25] .Histological findings of the whey protein and M. oleifera gel-coated implant group showed that the newly formed bone tissue was surrounded by a narrow connective tissue containing woven bone islets, blood capillaries, collagenous fibers, and a small number of inflammatory cells.These findings are consistent with those of Al-Molla et al. [16] .
Based on our literature survey, the anti-inflammatory and antibacterial properties of whey protein and M. oleifera gel are attributed to their lactoferrin, lactoglobulin, and lactalbumin contents.These contents could produce a higher number of osteoblastic cells and osteocytes in the experimental group than in the control group at the end of the 2 nd week [26] .Sixth weeks after surgery, we observed the thickening and increasing number of bone trabeculae in the implant area in the experimental group.However, only a few newly formed bone trabeculae were found in the control group.These findings indicate that healing and bone formation accelerate in the experimental animals than in the control animals.In this regard, Davison et al. reported similar results [26] .Sixth weeks after implantation, in the Iran.Biomed.J. 28 (2 & 3): 82-89 87 control group, the implant threads were filled with woven bone, whereas in the experimental group, they were filled with mature bone, characterized by osteon formation.These observations can be attributed to the antioxidant properties of whey protein [27] and M. oleifera gel [28] , as suggested by previous researchers [29][30][31][32][33] .Whey protein and M. oleifera extracts are thought to be superior sources for free cysteine, which boosts the production of glutathione and regulates bone cell differentiation while accelerating bone formation and decreasing bone marrow volume.At the end of the 6 th week after surgery in the experimental group, the bone marrow area significantly reduced compared to the control group, which supports the results obtained by Mohamed et al. [34] .
In the present study, IGF-I was detected by immunohistochemical method because osteocytes express significant amounts of IGF-I [35] , and disruption of the IGF-I gene in osteocytes impairs the development and growth of the bone.IGF-I is an important bone formation marker, related to PTH [36] , and is a signaling mediator of PTH receptor in the regulation of osteocytes during bone formation [37] .IGF-I is expressed in the proliferating osteoblasts and osteoblast-like cells in the early phase of bone formation [38] .In our study, collagen synthesis, osteoblastic activity, and IGF-I scores of osteoblastic cells, osteocytes, and osteoclasts in the experimental group reached the highest level at the end of the 2 nd week, while all scores of the control group were low.At the end of the second postoperative week, the healing rate and bone formation in the experimental group were improved, and the osteocyte, osteoblast, and osteoclast levels at the implant site were found to be higher than those in the control group.These results are consistent with Duan et al.'s findings [39] .
Type I collagenous fibers abundant in the osteoid tissue have been shown in Masson's trichrome-stained sections [40] .At the end of the 2 nd week of our experiment, the observation of randomly oriented collagen fibers and a few osteocytes indicated the presence of osteoid tissue in the area and also the start of the formation of mature bone tissue.At this period, abundant collagen fibers were observed in the defect area filled with fibrocartilage tissue in the experimental group.At the end of day six, the defect area in the experimental group was filled with mature bone with little collagen; however, in the control group, it was largely filled with woven bone and osteoid tissue, which supports the findings of Shayegan et al. [41] .

CONCLUSION
The pattern and progression of intramembranous osteogenesis can be observed by bone healing around the implant.In the implant area, fibrocartilage, osteoid tissue, woven bone, and lamellar bone are formed, respectively.As shown in this study, surface-treated implants with a mixture of whey protein and M. oleifera gel are well tolerated biologically.Due to earlier differentiation and proliferation of bone-forming cells, osseointegration is augmented by the new bone formation and mineralization that is accelerated and achieved on the surface treated implant sites.

Fig. 3 .
Fig. 3. IGF-I immunohistochemical staining of the sections of the defect sites in the (A) experimental and (B) control groups at the end of 2 nd week of the experiment.(A) Positive reactions of the osteoblasts, osteoclasts, and osteocytes are definite in the experimental group.(B) Negative staining of the osteocytes and stromal cells is clear in the control group.The sections were observed under a light microscope with magnitudes ×200 and ×400.

Fig. 4 .
Fig. 4. Sections of the defect sites in the study groups at the end of (A) 2 nd and (B) 6 th weeks.(A) Collagenous fiber intensities are seen in the fibrocartilage tissue.(B) Collagen staining intensity is higher in the control than in the experimental group.Masson's trichrome stain.The sections were observed under a light microscope with magnitudes ×200 and ×400.

Table 1 .
Histomorphometric results of the study groups at the end of the 2 nd and 6 th week of the experiment The differences between the mean values in the same line are statistically different (p < 0.01).

Table 2 .
Results of the immunohistochemical investigations at the end of the 2 nd and 6 th weeks